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Related Concept Videos

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Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
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Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...
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The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...
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CRISPR stands for Clustered Regularly Interspaced Short Palindromic Repeats is a adaptive immune system found in bacteria and archaea that protects against viral infections. This system enables prokaryotic cells to identify, remember, and neutralize foreign genetic elements, primarily bacteriophages, by storing fragments of the invader’s DNA as a genetic memory.The CRISPR immune response begins during an initial infection. Cas (CRISPR-associated) proteins play a central role in this...
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Related Experiment Video

Updated: Sep 13, 2025

Field-Deployable Candidatus Liberibacter asiaticus Detection Using Recombinase Polymerase Amplification Combined with CRISPR-Cas12a
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cliCRISPR: crRNA-limited CRISPR/Cas12a system for multiplexed detection.

Bo Yan1, Cong Wei2, Xueying Lei2

  • 1Department of stomatology, The First Affiliated Hospital of Zhengzhou University, Zhengzhou, 450052, China.

Biosensors & Bioelectronics
|August 1, 2025
PubMed
Summary
This summary is machine-generated.

We developed cliCRISPR, a novel CRISPR/Cas12a method using limited crRNA for multiplexed nucleic acid detection. This strategy correlates fluorescence intensity with crRNA concentration, enabling precise genotyping and biosensor development.

Keywords:
CRISPR/Cas12aNucleic acid analysisSingle nucleotide polymorphismVitamin D

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Genetics

Background:

  • CRISPR/Cas12a is widely used for nucleic acid detection, typically requiring excess crRNA.
  • Non-specific trans-cleavage activity of CRISPR/Cas12a hinders multiplexed detection in single-pot assays.
  • Existing methods face challenges in achieving sensitive and specific multiplexed detection.

Purpose of the Study:

  • To introduce a crRNA-limited strategy, cliCRISPR, for multiplexed detection using the CRISPR/Cas12a system.
  • To demonstrate that fluorescence intensity can be correlated with crRNA concentration for multiplexed quantification.
  • To validate the cliCRISPR method for precise genotyping of genetic variations.

Main Methods:

  • Developed a crRNA-limited CRISPR/Cas12a strategy (cliCRISPR).
  • Correlated fluorescence intensity with specific crRNA concentrations for distinct signal levels.
  • Applied a logic-gate strategy for genotyping rs4646536, a variant linked to vitamin D deficiency.
  • Validated the method using clinical samples and compared results with TaqMan qPCR.

Main Results:

  • Achieved distinguishable fluorescence intensities using controlled crRNA concentrations (10 nM crRNA1, 3 nM crRNA2).
  • Successfully genotyped rs4646536 (wild-type, mutant, heterozygous) with high accuracy.
  • Demonstrated consistency with TaqMan qPCR results and adherence to Mendelian inheritance patterns.
  • Validated the practicability of cliCRISPR for multiplexed genotyping in family samples.

Conclusions:

  • cliCRISPR offers a novel crRNA-limited approach for multiplexed nucleic acid detection.
  • The method enables precise genotyping by correlating fluorescence with crRNA concentration.
  • cliCRISPR shows significant potential for developing advanced multiplexed biosensors based on CRISPR/Cas12a technology.